Yang Yining

e0125 MMP-9 gene polymorphisms contribute to coronary artery disease risk in the Uighur population of China

Background Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in early atherosclerosis, vascular remodelling and development of atherosclerotic lesion. The potentially functional MMP-9 polymorphisms may contribute to the susceptibility of coronary artery disease (CAD). We aimed to investigate the association between three SNPs (−1562C>T, R279Q, R668Q) of the MMP-9 gene with CAD in the Uighur population of China. Materials and methods 375 angiographic ally proven patients with coronary artery disease and 417 sex-matched and ethnically matched controls were genotyped for MMP-9 polymorphisms by the PCR-restriction fragment length polymorphism (PCR-RFLP) technique. Genotype/allele frequencies were compared in patients and controls using the χ2 test. The relationship between the polymorphism of the MMP-9 gene and the severity of coronary arterial stenosis was analysed also. Results At MMP-9 -1562 locus, there were significant differences between patients and controls (p<0.05), leading to significant OR for TT genotype (OR=2.93, CI 1.03 to 8.72) and R allele (OR=1.85, CI 1.34 to 2.54). These OR were higher in the sub-sample of smokers (3.87 and 2.06, respectively). Binary logistic regression analysis also confirmed that R allele carriers (CT and TT) have a higher risk of CAD (OR=2.07, CI 1.09 to 2.95). MMP-9 R279Q locus did not show significant differences between patients and controls. But QQ genotype and Q allele were significant risk factors in the smoker group. Q allele carriers (RQ and QQ) were also significantly associated with CAD risk in the smoker group (OR=1.43, CI 1.13 to 1.2.26). The R668Q locus did not show significant differences between two groups. And the MMP-9 polymorphism may not be useful as a predictor of the severity of coronary atherosclerosis. Conclusions MMP-9 -1562T allele and TT genotype are significantly associated with CAD patients from the Uighur Population of China (Xinjiang). This association was stronger in smokers, supporting the conclusion that an interaction between MMP-9 activity and smoking augments CAD risk. Further studies with larger sample size are warranted to confirm these associations in different populations.

e0228 Transfection of recombinant adeno-associated virus serotype 9 to mouse heart in vivo and the effects on cardiac function

Objective To evaluate the transfection efficiency of recombinant adeno- associated virus serotype 9 carrying enhanced green fluorescent protein (rAAV9- eGFP) to mouse heart in vivo and the effects on cardiac function. Methods 1. 16 C57BL/6 mice were transfected rAAV9-eGFP by tail injection. EGFP expression in the heart, liver, lung, kidney and brain cryosections was observed under inverted fluorescence microscope 7, 14, 21, 28 days after the injection of rAAV9-eGFP and eGFP was quantitated by Western Blot. 2. 20 C57BL/6 mice were divided into control group and rAAV9-eGFP group randomly, and were received with saline or rAAV9-eGFP. The echocardiography and haemodynamics were performed 28 days after the injection of saline or rAAV9-EGFP. Results 1. EGFP expression in the heart reached the maximum at day 21, at the point of which the transduction efficiency of rAAV9-eGFP in myocardium was 32%. The other tissues had a little or no eGFP expression. 2. The cardiac function did not reveal significant difference between rAAV9-eGFP group and the control group after transfection (p>0.05). Conclusion rAAV9-eGFP gene can be stably and efficiently expressed in mouse heart, and has no toxic effect on cardiac function.

ASSA13-13-3 Investigation of Prevalence and Distributing Feature of Chronic Heart Failure in Xin Jiang Adult Population

Objective To investigate the prevalence and distributing feature of chronic heart failure (CHF) in Xin jiang adult population. Methods Four-stage selected random samples Maternal Age 35 and over were used to analyse the prevalence and distributing feature of self-reported congestive heart failure in different nationality, age, sex and The sampled adult population were collected 6 locality (Urumqi, Ke lamayi, Fu kang, the Turfan Basin locality, He tian locality, Yi li Hazakh autonomous prefecture) from 23 municipalities and 7 locality and 5 autonomous county in Xin jiang. The proportion of male to female accounted for 50% each. Results A total of 8459 adults were surveyed. The prevalence of CHF was 1.26%, The prevalence of CHF was 0.89%, 1.11% and 2.14% in Han, Uygur and Hazakh population, respectively. The risk of CHF was higher in Hazakh than Han and Uygur people. 1.61% and 0.93% for the general population, the males and females, respectively. The males had CHF more frequently than the females (u = 2.79, P < 0.05). The prevalence of CHF was 0.29%, 0.60%, 1.32%, 2.55% and 4.10% in 35–44, 45–54, 55–64, 65–74, 75 and over years of age groups, respectively. Therisk of CHF increased substantially with ageing. Common complications of CHF was hypertension (63.55%), coronary heart disease (42.99%), diabetes (18.69%), valvular heart disease (5.61%) and fibrillation atrial (4.67%). Conclusions The results of this study showed that the higher prevalence of CHF in Xinjiang. The prevalence of CHF increased substantially with ageing and difference between nationality. Investigation of prevalence and distributing feature of chronic heart failure, research the prevention and treatment of CHF among high-risk population and using proper measures of intervention to decrease the prevalence of CHF among population is necessary in Xin jiang.

ASSA13-11-7 Distributional Characteristics of Genetic Polymorphisms of Serum Amyloid Protein A1 in Healthy Chinese Han and Uighur Population in Xinjiang

Objective To explore the distributional characteristics of genetic polymorphisms (rs2229338 and rs12218) of serum amyloid protein A1 (SAA1) in healthy Chinese Han and Uighur population of Xinjiang. Methods 316 Uighur and 362 Han healthy persons were detected the genotypes of the SAA1 by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results The genotype distributions of the Uighur group and the Han group were in the Hardy-Weinberg equilibrium (both P > 0.05). The frequencies of AA, AG and GG of rs2229338 were 76.6%, 23.4%, and 0 in Uighur group while ones were 91.7%, 7.7% and 0.6% in Han group. There was significant difference in distribution of genotypes between these two groups (P < 0.001). The frequencies of CC, CT and TT of rs12218 were 10.1%, 47.5%, and 42.4% in Uighur group while ones were 3.3%, 34.3% and 62.4% in Han group. There was also significant difference in distribution of genotypes between these two groups (P < 0.001). The A-C and G-T haplotypes were more frequency in the Uighur but the A-T haplotype was more common in the Han population, respectively (both P < 0.001). Conclusions The mutational frequencies of the tagging SNP (rs2229338 and rs12218) in the SAA1 gene of Uighur population were higher than those of Han.

ASSA13-03-2 Distributional Characteristics of Apolipoprotein A5 Gene –1131 T > C

Objective To explore the distributional characteristics of Apolipoprotein A5 (ApoA5) Gene –1131 T > C Polymorphism and the influence of serum lipid in Chinese Han and Uighur population of Xinjiang. Methods 416 Uighur and 525 Han persons were detected the genotypes of the ApoA5 Gene –1131 T > C Polymorphism by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results The frequencies of TT, TC and CC genotype of ApoA5 gene –1131 T > C were 35.1%, 53.7% and 11.2% in Uighur group while same genotypes were 43.7%, 46.3% and 10% in Han group. There was not statistics difference in distribution of genotypes between these two groups (P > 0.05). In the two groups individuals carried C allele (TC+CC genotype) had higher level of serum triglyceride than those carried TT genotype. No-condition Logistic regression analyses adjusted for age, gender, smoking, dringking, BMI and serum total cholesterol, revealed that individuals in the two groups who carried C allele had an increased risk of HTG compared with TT genotype (OR = 2.81, 95%CI: 1.54 ∼ 5.35 in Uighur group; OR = 3.18, 95%CI: 1.81 ∼ 5.74 in han group). Conclusions The mutational frequencies of the –1131 T > C polymorphism of ApoA5 gene in Uighur population were not statistics difference with Han. The polymorphism may be associated with level of serum triglyceride in Uighur and Han population of Xinjiang and T allele might be a risk factor of hypertriglyceridemia.

ASSA13-03-23 Effects Caused by Postconditioning of Mouse Heart Suffering from Reperfusion Injury and Mechanisms Induced by Reperfusion Injury Salvage Kinase

Objective Explore cardioprotective mechanisms induced by reperfusion injury salvage kinase (ERK1/2) signal transduction pathways changes in isolated mouse hearts. Methods Established Langendorff perfused mouse heart model, 96 C57BL mouse hearts were divided to 4 groups: 1) I/R control: 30 min global ischemia and 2 h reperfusion; 2) IPC:IPC with 3 episodes of 10 s of ischemia and 10 s reperfusion after 30 min ischemia and before 2 h reperfusion; 3) IPC+ERK1/2 inhibitor PD98059 (10–5 mol/L for 15 min); 4) I/R+ERK1/2 inhibitor PD98059 (10–5 mol/L for 15 min). The effects of IPC on Ischemic size, myocardial cell apoptosis index, phospho-protein kinase (P-ERK1/2) and phospho-protein kinase B(P-Akt) were measured. Expressions of apoptosis related proteins, including Bax, Bcl-2 and Cyt.C, in cytosolic and membrane fraction were detected by Western blot. Results Following 180min reperfusion, infarct size was significantly reduced in postconditioning group (32.8 ± 3. 8)% compared with I/R group hearts [(48.1 ± 4.8)%, P < 0.05], and equally improved myocardial function compared with I/R group (all P < 0.05). Compared with ischemia reperfusion group, ischemic postconditioning group markedly decreased apoptotic index (AI)(P < 0.05), increased the mRNA expression of Bcl-2 gene (P < 0.05), the ratio of Bcl-2 to Bax was incidentally up-regulated (P < 0.05). Although IPC did not induce changes in the level of Bcl-2 expression in cytosolic fraction compared with I/R groups, the expression of Bcl-2 inmembrane fraction was up regulatedin IPC group compared with I/R group. The increase in the lease of mitochondrial Cyt.C into cytosol induced by I/R was significantly reduced by IPC. The histopathological changes in mitochondria and myocardium destroyed by ischemia repefusion were alleviated markedly by ischemic postconditioning. IPC significantly increased myocardial ERK1/2 phosphorylation, PD98059 inhibited the phosphorylation of ERK1/2 and abolished the cardioprotective effects induced by IPC. Conclusions Postconditioning plays a pivotal role in reducing myocardial infarct size after ischemia/reperfusion injury. IPC right before reperfusion but not after reperfusion attenuated I/R injury of isolated mouse hearts via the upstream target, ERK1/2-MAPK signal transduction pathway which mediated the expression of Bcl-2/Bax and the release of mitochondrial Cyt.C.

ASSA13-11-2 Relationship Between Novel Polymorphisms of the C5L2 Gene and Coronary Artery Disease

Objective C5L2, a G protein-coupled 7-transmembrane domain complement, has been demonstrated to be a functional receptor of acylation-stimulating protein (ASP), which is a stimulator of triglyceride synthesis or glucose transport. In this study, we will investingate the variations in the coding region of the C5L2 gene (C698T and G901A) and their association with coronary artery disease (CAD). Methods We identified novel single nucleotide polymorphisms (SNPs), (C698T and G901A) in exon 2 using a polymerase chain reaction direct-sequencing method. We examined the role of this SNP for CAD using two independent case-control studies: one was in the Han population (492 CAD patients and 577 control subjects) and the other was in the Uygur population (319 CAD patients and 554 control subjects). Results Using a polymerase chain reaction direct-sequencing method. We identified novel SNPs, 698C > T (P233L), and G901A (A300H) in exon 2. Heterozygote carriers of the 698CT genotype were more frequent among CAD patients than among controls not only in the Han population (7.3% versus 1.7%) but also in the Uygur population (4.7% versus 1.6%). The odds ratio (OR) for carriers of the 698CT genotype for CAD was 4.484 (95% confidence interval (CI): 2.197–9.174) in the Han group and 2.989 (95% CI: 1.292–6.909) in the Uygur population. After adjustment of confounding factors such as sex, age, smoking, alcohol consumption, hypertension, diabetes, as well as serum levels of triglyceride, total cholesterol, high-density lipoprotein, the difference remained significant in the Han group (OR = 6.604, 95% CI: 2.776–15.711, P < 0.001) and in the Uygur group (OR = 2.602, 95% CI: 1.015–6.671, P = 0.047). Heterozygote carriers of the 901GA genotype were more frequent among controls than among CAD patients not only in the Han population (8.6% versus 1.8%) but also in the Uygur population (5.2% versus 0.9%). The OR for carriers of the 901GA genotype for CAD in the Han population was 0.205 and 0.172 in the Uygur population. After adjustment of confounding factors, the difference remained significant in the Han group (OR = 0.143, 95% CI: 0.068–0.302, P < 0.001) and in the Uygur group (OR = 0.246, 95% CI: 0.072–0.837, P < 0.001).Two haplotypes in the Han population in the result analysis, which the C-G haplotype frequencies in the CHD group was significantly lower than the control subjects (χ2 = 22.713, P < 0.001), the T-G haplotype distribution in the the CHD group was significantly higher (χ2 = 19.022, P < 0.001). In Uighur population, analysis of the T-G haplotypes frequency in case group and the control group was (0.025 vs 0.008, P = 0.004); C-A haplotypefrequency in CAD group and the control group (0.026 vs 0.005, P = 0.001). Conclusions (1) C698T and the G901A in Han and Uygur population are association with coronary heart disease; (2) The 698CT and 901GA genotype of C5L2 may be a genetic maker of CAD in the Han and Uygur population in western China.

Association of the rs7257062T/C of ASP gene polymorphism and coronary heart disease

Objective To investigate the association of the rs7257062T/C polymorphism of ASP gene with coronary heart disease (CHD) and the influence of serum lipid levels in Chinese Han population patients of Xinjiang. Methods The polymorphism of gene in 425 patients with CHD and 439 controls was analysed by polymerase chain reaction restriction fragment length polymorphism; the serum lipid levels were detected as well. Results The frequencies of TT, TC and CC genotype were 70.1%, 25.60% and 4.2 % in the CHD group, while they were 48.3%, 42.8 % and 8.9 % in the control group. There was a significant difference in the distribution of genotypes between the two groups (p<0.01). Logistic regression analyses adjusted for age, gender, smoking, serum total cholesterol, presence of hypertension and diabetes revealed that individuals carrying CC genotype had an increased risk of CHD compared with TT genotype (OR=0.351, 95% CI: 0.172∼0.716). There was also a significant difference in serum triglyceride level in genotypes between these two groups (P). Patients in CHD group who carried CC and TC genotypes had lower serum triglyceride level than the TT genotype carriers. Conclusion The rs7257062T/C polymorphism of ASP gene has an influence on the serum triglyceride level in Han population of Xinjiang. This polymorphism might be associated with development of CHD, and the CC genotype might be a protective factor in the development of CHD.

Analysis of differences of expression efficiency about rAAV9 vector with different promoters in rats myocardial cells

Objective To compare the expression efficiency and the effect on cell growth about recombinant adeno-associated serotype 9 vector with different promoters in H9C2 cells. We aim to examine whether it can be a foundation for further research of rAAV9 vector used for the gene therapy of ischemic heart disease. Methods rAAV9-CMV-eGFP and rAAV9-CBA-eGFP were transfected into H9C2 cells at same multiplicities of infection (MOI=1×107), respectively. Enhanced green fluorescent protein (eGFP) expression in the cells was observed under inverted fluorescence microscope, and the eGFP-positive cell percentage determined by flow cytometry. The expression of eGFP was quqntitated by Western blot. Alamar Blue assay was used to assess the proliferation of the transfected cells. Results The transduction efficiency of rAAV9-CBA-eGFP and rAAV9-CMV-eGFP in H9C2 cells was (92.5±1.0)% and (74.2±1.3)% (p<0.05), respectively. eGFP expression of rAAV9-CBA-eGFP is stronger than rAAV9-CMV-eGFP. Alamar Blue assay did not reveal significant differences in the absorbance between the transfected cells and the control cells after transfection. Conclusion The CBA promoter directs the higher expression from an rAAV9 vector than the CMV promoter in the H9C2 cells without causing cell growth inhibition.

Relationship between a novel polymorphism of C5L2 gene and coronary artery disease in Chinese Han and Uyghur population

Background C5L2 was demonstrated a functional receptor of acylation-stimul- ating protein (ASP) which is a stimulator of either triglyceride synthesis or glucose transport. However, little is known about the variations in the coding region of the C5L2 gene and theirs association with coronary artery disease (CAD). Methodology/principal findings In the present study, we identified a novel single nucleotide polymorphism (SNP), 698 C>T (P233L), in exon 2 using PCR-directly sequencing method. This nucleotide change causes the amino acid change from proline to leucine at codon 233. We examined the role of this SNP for CAD using a 2-stage design, first we determined the prevalence of C5L2 genotypes in 492 CAD patients and 577 age and sex-matched healthy control subjects of Han population, Second, a replication analysis of 319 CAD patients and 554 ageand sex-matched healthy control subjects of Uyghur population. Heterozygote carriers of the 698CT genotype were more frequent among CAD patients than among controls in Han (7.3% vs 1.7%) and in Uyghur population (4.7% vs 1.6%), respectively. The OR for carriers of this genotype for CAD was 4.484 (95% CI 2.197 to 9.174) in Han and 2.989 (95% CI 1.292 to 6.909) in Uyghur population, respectively. After adjustment of the confound factors such as sex, age, smoking, drinking, hypertension, diabetes, triglyceride, total cholesterol, high density lipoprotein, the difference remains significant both in Han (p<0.001, OR 6.604, 95% CI 2.776 to 15.711) and in Uyghur population (p=0.047, OR 2.602, 95% CI 1.015 to 6.671), respectively. Conclusion/significance We conclude that the 698CT genotype of C5L2 may be a genetic maker of CAD in Chinese Han and Uyghur population.